A crucial early step in mounting an inflammatory response is the passage of leukocytes from the bloodstream to the injured tissue. To reach the affected site, the leukocytes must breach the endothelium lining the vessel wall, the underlying basement membrane, and the surrounding connective tissue. Little is known about the mechanisms that control leukocyte adhesion to and migration across endothelium, largely due to lack of appropriate model systems. The proposed research will use monolayers of endothelial cells cultured on connective tissue derived from human amnion as in vitro models of the vascular wall. Isolated human polymorphonuclear leukocytes (PMNs) placed above such cultures adhere to and migrate across the endothelial monolayers in response to gradients of chemotactic agents. This in vitro system will be used to examine several fundamental questions regarding leukocyte transendothelial migration. Stimulated leukocytes secrete a variety of degradative enzymes; the role of these enzymes in aiding PMN migration will be studied using specific inhibitors and antibodies. Possible modulation of PMN migration by other components of the blood, including plasma, lymphocytes, monocytes, platelets, and red cells, will be examined. To determine the specificity of the interaction between leukocytes and endothelium, migration of PMNs across monolayers of endothelial cells derived from different species and from different parts of the vascular tree will be compared. In addition, to assess the importance of the endothelial intercellular junction as a barrier to leukocyte penetration, PMN migration across endothelial monolayers with high or low electrical resistance will be compared. Lastly, cell surface macromolecules that are involved in the interactions between PMNs and endothelium will be identified using monoclonal antibodies. These studies promise to provide new information about the molecular mechanisms that govern leukocyte extravasation during the normal inflammatory response. Such knowledge regarding this critical step in inflammation also may aid in understanding related phenomena, including recirculation of lymphocytes, invasion of blood-borne pathogens, and metastasis of tumor cells.